Separation agent for separating optical isomer and method for preparation thereof

ABSTRACT

A method of producing a novel separating agent for separating enantiomeric isomers, characterized by including: a step of supporting an optically active polymer compound and a compound having an asymmetric structure of a molecular weight of 1,000 or less on a carrier using a solvent; a step of removing the solvent; and a step of removing the compound having the asymmetric structure of the molecular weight of 1,000 or less by washing.

This is a division of Ser. No. 10/480,398, filed Dec. 9, 2003 now U.S.Pat. No. 7,090,775, which was the national stage of InternationalApplication No. PCT/JP02/06853, filed Jul. 5, 2002, which InternationalApplication was not published in English.

TECHNICAL FIELD

The present invention relates to a novel separating agent for separatingenantiomeric isomers, a method of producing the same, and a separationmethod for enantiometric isomers using the same, in particular, aseparation method for enantiomeric isomers with chromatography. Thepresent invention provides a separation technique of the enantiomericisomers, which enable the optical resolution of chiral compounds with ahigh separation coefficient in analyses of medicines, foods,agricultural chemicals, fragrant materials, and the like.

BACKGROUND ART

Many organic compounds have enantiomeric isomers which have completelythe same physical and chemical properties but differ in physiologicalactivity. This is attributable to the following. In most cases, proteinsor glucides per se constituting a living being are composed of only oneof the enantiomeric isomers, and for this reason, a difference arisingin the manner of acting on the other enantiomeric isomer results in adifference in physiological activity. In particular, in many cases,there are significant differences in medical properties and toxicitybetween the enantiomeric isomers of the pharmaceuticals, and thisproblem is recognized as a significant problem in the field ofpharmaceuticals. The Ministry of Health, Labour and Welfare of Japanprescribes in the Guideline for the Production of Medicines that “in thecase where the drug is a racemi form, it is desirable that studies onthe dynamic behaviors of absorption, distribution, metabolism andexcretion be made on each enantiomeric isomer”.

Since the enantiomeric isomers have completely the same physical andchemical properties, for example, the physical properties such as aboiling point, a melting point or a solubility, as described above, theycannot be analyzed by ordinary separation means. For this reason,extensive investigations have been made on techniques for analyzing awide variety of enantiomeric isomers in a simple manner and with a highprecision. As a result, an optical resolution method by high performanceliquid chromatography (HPLC), in particular, an optical resolutionmethod using an enantiomeric isomer separation column for HPLC has beendeveloped as an analyzing method that meets those requirements. Theenantiomeric isomer separation column defined herein uses an asymmetryidentifying agent itself or a chiral immobilizing phase having theasymmetry identifying agent supported on a suitable carrier.

As examples of the chiral immobilizing phase, optically activepoly(triphenylmethyl methacrylate). (cf., JP-A 57-150432), cellulose andamylose derivatives (cf., JP-A 60-40952, JP-A 60-108751, JP-A 60-142930and JP-A 63-178101), ovomucoid, which is a protein (JP-A 63-307829), andthe like have been developed. It is known that, of the many chiralimmobilizing phases for HPLC, a column for enantiomeric isomerseparation having the cellulose or amylose derivatives supported onsilica gel has a high asymmetry identification ability for a very widevariety of compounds (for example, Okamoto et al., Angew. Chem. Int.Ed., 1998, 37, 1020).

In the case of aiming at analyses such as an optical purity measurement,it has been desired that as many unidentified enantiomeric isomercompounds as possible can be separated by as few kinds as possible ofenantiomeric isomer separation columns. As a result, the above-mentionedcolumn for enantiomeric isomer separation having the cellulose oramylose derivatives supported on the silica gel has been accepted aspractical separation media.

In recent years, studies on liquid preparative chromatography foroptically active substances on an industrial scale in a combination of achiral immobilizing phase for HPLC and a simulated moving bed methodhave been developed (Phrarm. Tech. Japan, 12, 43(1996). In such studies,not only analysis, but also preparative separation, namely,chromatographic separation as a production means, are noted.

For that purpose, in order to not only merely perform base lineseparation, but to improve the productivity of the preparativechromatography and decrease the production cost, it has been demanded todevelop a chiral immobilizing phase that enables the further separationof a target compound for the limited, specified preparative separation,that is, has a value of separation coefficient α as high as possible.

On the other hand, a molecular imprinting method is known as a method ofspecifically identifying the specific target compound. The most popularmethod in general production methods for a molecular template is thatthe target compound (guest) and a monomer for performingnon-conjugated-bond-type interaction therewith are reacted in a testtube using a crosslinking agent or the like for polymerization, therebyobtaining a polymer compound (host). Further, a method of obtaining ahost by mixing the guest and a polymer and subjecting the polymer to acrosslinking reaction in an interaction state is known (for example, G.Wulff et al., Angew. Chem., 1972, 84, 364).

A filler for chromatography obtained by such molecular imprinting has ahigh identification ability for a guest. However, it is known that sucha filler is extremely high in its degree of adsorption for a guest, andas a result, the corresponding elution peak shows a widely extendedform. This is not satisfactory in terms of chromatography efficiency.Further, the polymer compound obtained by the molecular imprintingmethod cannot include a dissolution operation that may decompose aprepared template. For this reason, the polymer compound is preparedinto the filler for chromatography by pulverization processing or thelike. However, an operation such as classification is complicated, and aparticle size is not uniform, resulting in a decrease in chromatographyefficiency. For the above reason, the filler for chromatography obtainedby the technique using the molecular imprinting method has not been yetput into practical use.

A purpose of the present invention is to provide a process for producinga novel separating agent for separating enantiomeric isomers to obtain aseparating agent for separating enantiomeric isomers, which is greatlyimproved in separation efficiency for objective compounds forseparation.

Another purpose of the present invention is to provide a separatingagent for separating enantiomeric isomers, which has a greatly improvedseparation performance for a compound to be separated and can separatecompounds that could not conventionally be separated.

Still another purpose of the present invention is to provide animmobilizing phase for chromatography or an immobilizing phase forcontinuous liquid preparative chromatography, using the separating agentfor separating enantiomeric isomers, and a separation method forenantiomeric isomers.

DISCLOSURE OF THE INVENTION

The present inventors have found that a separation ability is greatlyimproved by improving a separating agent for separating enantiomericisomers, in place of selective use of a general-purpose developingsolvent conventionally used, specifically improving the separating agentfor separating enantiomeric isomers using a compound having anasymmetric structure of a molecular weight of 1,000 or less, as a methodof increasing the separation ability of the separating agent forseparating enantiomeric isomers.

More specifically, according to the present invention, there is provideda method of producing a novel separating agent for separatingenantiomeric isomers, characterized by including the addition of acompound having an asymmetric structure of a molecular weight of 1,000or less in supporting an optically active polymer compound on a carrier.

Further, according to the present invention, there is provided a methodof producing a novel separating agent for separating enantiomericisomers, characterized by including: a step of supporting an opticallyactive polymer compound and a compound having an asymmetric structure ofa molecular weight of 1,000 or less on a carrier using a solvent; and astep of removing the solvent.

Further, according to the present invention, there is provided a methodof producing a novel separating agent for separating enantiomericisomers, characterized by including: a step of supporting an opticallyactive polymer compound and a compound having an asymmetric structure ofa molecular weight of 1,000 or less on a carrier using a solvent; a stepof removing the solvent; and a step of removing the compound having theasymmetric structure of the molecular weight of 1,000 or less bywashing.

Further, according to the present invention, there is provided a methodof producing a novel separating agent for separating enantiomericisomers, characterized by including: a step of supporting an opticallyactive polymer compound on a carrier using a solvent; a step ofadditionally supporting a compound having an asymmetric structure of amolecular weight of 1,000 or less on the carrier; and a step of removingthe solvent.

Further, according to the present invention, there is provided a methodof producing a novel separating agent for separating enantiomericisomers, characterized by including: a step of supporting an opticallyactive polymer compound on a carrier using a solvent; a step ofadditionally supporting a compound having an asymmetric structure of amolecular weight of 1,000 or less on the carrier; a step of removing thesolvent; and a step of removing the compound having the asymmetricstructure of the molecular weight of 1,000 or less by washing.

Further, according to the present invention, there is provided aseparating agent for separating enantiomeric isomers, including anoptically active polymer compound supported on a carrier, from which acompound having an asymmetric structure of a molecular weight of 1,000or less added as a production raw material and supported is removed.

Further, according to the present invention, there is provided animmobilizing phase for chromatography using the separating agent forseparating enantiomeric isomers or an immobilizing phase for acontinuous liquid preparative chromatography using the separating agentfor separating enantiomeric isomers or a separation method forenantiomeric isomers using the separating agent for separatingenantiomeric isomers.

BEST MODE FOR CARRYING OUT THE INVENTION

A method of producing a novel separating agent for separatingenantiomeric isomers of the present invention is described.

The production method of the present invention includes a step of addinga compound having an asymmetric structure of a molecular weight of 1,000or less in supporting an optically active polymer compound on a carrier,and a production method including the following steps can be exemplifiedas the production method including the above step.

Firstly, in a first step, an optically active polymer compound and acompound having an asymmetric structure of a molecular weight of 1,000or less are supported on a carrier using a solvent. Here, in the presentinvention, the optically active polymer compound is directly supportedon the carrier. However, the compound having the asymmetric structure ofthe molecular weight of 1,000 or less is not directly supported on thecarrier, but is indirectly supported on the carrier by physically orchemically bonding it to the optically active polymer compound.

In this step, the following methods can be applied:

(1) a method of preparing a solvent solution of an optically activepolymer compound and the compound having the asymmetric structure of themolecular weight of 1,000 or less, and bringing the solution and thecarrier in contact with each other by a method of immersing the carrierin the solution, a method of applying the solution to the carrier, orthe like, thereby supporting it on the carrier; and

(2) a method of preparing the solvent solution of the optically activepolymer compound, contact-supporting the solution on the carrier by amethod of immersing the carrier in the solution, and dissolving thecompound having the asymmetric structure of the molecular weight of1,000 or less in the solution, or after preparation of the solventsolution, adding the solvent solution of the compound having theasymmetric structure of the molecular weight of 1,000 or less to thesolution, thereby supporting it on the carrier.

The term “supporting” used herein means that an optically active polymercompound and a compound having an asymmetric structure of a molecularweight of 1,000 or less are fixed to the carrier. This fixation isperformed by physical adsorption and/or chemical bonding between theoptically active polymer compound and a compound having an asymmetricstructure of a molecular weight of 1,000 or less, and a carrier.

The physical adsorption means that the compound is adsorbed on a surfaceof the carrier and/or inside fine pores of the carrier.

The chemical bonding includes bonding between a carrier and an opticallyactive polymer compound, bonding between a part of the optically activepolymer compound physically adsorbed on the carrier, bonding betweenremaining optically active polymer compound and the compound having theasymmetric structure of the molecular weight of 1,000 or less, andchemical bonding between the carrier and the optically active polymercompound by reaction with a crosslinking agent, reaction with a radicalgenerator or light irradiation (irradiation with radiation such asγ-rays, or irradiation with electromagnetic waves such as microwaves).

When chemically bonding the carrier and the optically active polymercompound, it is desirable to perform chemical bonding before or after astep of removing the compound having the asymmetric structure of themolecular weight of 1,000 or less as a post-step.

Examples of the optically active polymer compound used in the presentinvention include polymers or copolymers of (meth)acrylates or(meth)acrylamides that do not have optically active substituents,(meth)acrylates or (meth)acrylamides having the optically activesubstituents, styrene, acetylene or the like, polysaccharides or theirderivatives, peptides, and proteins.

Of those, the polymer compounds having the asymmetry identificationability for a compound to be separated are preferable. In particular,the polymers or copolymers of (meth)acrylates or (meth)acrylamides,polysaccharides and their derivatives, and proteins that are known tohave the asymmetry identification ability are preferable, polymers orcopolymers of (meth)acrylamides or (meth)acrylates, polysaccharides andtheir derivatives having optically active substituents at side chainsare more preferable, and polysaccharide derivatives are most preferable.

Note that, the (meth)acrylates used in this specification mean acrylatesand methacrylates, and the (meth)acrylamides used in this specificationmean acrylamides and methacrylamides.

As a polysaccharide, any synthetic polysaccharide, any naturalpolysaccharide, and any modified natural polysaccharide may be used solong as they have an optical activity. Those which have a highregularity in the binding form are more desired.

There are exemplified β-1,4-glucan (cellulose), α-1,4-glucan (amylose,amylopectin), α-1,6-glucan (dextran), β-1,6-glucan (busturan),β-1,3-glucan (for example, curdlan, schizophyllan, etc.), α-1,3-glucan,β-1,2-glucan (Crown Gall polysaccharide), β-1,4-galactan, β-1,4-mannan,α-1,6-mannan, β-1,2-fructan (inulin), β-2,6-fructan (levan),β-1,4-xylan, β-1,3-xylan, β-1,4-chitosan, α-1,4-N-acetylchitosan(chitin), pullulan, agarose, alginic acid, and the like. Also, thepolysaccharide includes starch containing amylose.

Among those, cellulose, amylose, β-1,4-xylan, β-1,4-chitosan, chitin,β-1,4-mannan, inulin, and curdlan, which are readily available as apolysaccharide having a high purity, are preferred. Cellulose andamylose are particularly preferred.

These polysaccharides have a number-average degree of polymerization (anaverage number of pyranose rings or furanose rings contained in onemolecule) of at least 5, preferably at least 10, or preferably 1,000 orless in view of ease of handling, though there is no particularlimitation in the upper limit thereof.

The polysaccharide derivative is a compound combined with a compoundhaving a functional group reactive with part or all of the hydroxylgroups of the polysaccharide through an ester bond, an urethane bond oran ether bond.

The compound having a functional group capable of reacting with ahydroxyl group can be any compound so long as it is a compound havingleaving groups such as substituted or unsubstituted aromatic, aliphaticor alicyclic carboxylic acids, acid halides, acid anhydrides, carboxylicacid derivatives such as acid ester, substituted or unsubstitutedaromatic, aliphatic or alicyclic isocyanic acid derivatives, alcohols,and other compounds. The compound may have or may not have opticallyactive groups.

Preferable polysaccharide derivatives are polysaccharide esterderivatives and carbamate derivatives, and polysaccharide esterderivatives and carbamate derivatives having 0.1 or more, per glucoseunit, of ester bond or urethane bond are particularly preferable.

The amount of the optically active polymer compound used is such anamount that the amount of the compound supported on a carrier withrespect to a carrier mass preferably corresponds to 1 to 100 mass %,more preferably 5 to 60 mass %, and most preferably 10 to 40 mass %.

The compound having the asymmetric structure of the molecular weight of1,000 or less used in the present invention includes the following (I)and (II).

(I) A compound to be separated in the case of being used as a separatingagent for separating enantiomeric isomers, or its similarly structuredcompound. The “similarly structured compound” used herein is a compoundin which a functional group is similar to that of the compound to beseparated, the number of methylene chains increases or decreases, thenumber of substituents increases or decreases, a position of thefunctional group differs, and a kind of functional group differs fromthe compound to be separated and which accordingly has such a structurethat a molecular size is, for example, larger by about 1 to 5 carbonatoms or smaller by about 1 to 5 carbon atoms. Examples of the similarlystructured compound, with respect to 1-phenyl-2-propanol, include1-phenylethanol, 2-phenyl-2-propanol, 1-phenyl-2-butanol,2-phenyl-2-butanol, 3-phenyl-2-butanol, 1-substituted phenyl-2-propanol,1-(1-naphthyl)-2-propanol, 1-(2-pyridyl)-2-propanol, and1-cyclohexyl-2-propanol. Examples of the similarly structured compound,with respect to 1-(1-naphthyl)-ethanol, include 1-(9-anthryl)-ethanol.

Besides, the “similarly structured compound” includes compounds in whichenvironmental conditions around the asymmetric carbon are similar.

(II) A compound to be separated in the case of being used as aseparating agent for separating enantiomeric isomers, or a compoundother than a similarly structured compound thereof, preferably acompound having a cyclic structure of a molecular weight of 40 to 1,000,preferably 60 to 600.

Preferable compounds (I) and (II) are compounds having a polarfunctional group such as a heteroatomic group, e.g., a hydroxyl group, acarbonyl group, an amino group or a carboxyl group, compounds having afunctional group related to π-electron interaction, such as a benzenering, racemi form (±), and optically active substance (+) or (−) arepreferable.

The compound (I) has a molecular weight of 40 or more, preferably 60 to1,000, and more preferably 100 to 500.

The compound having the asymmetric structure of the molecular weight of1,000 or less is used in an amount of preferably 0.01 to 1,000 mass %,more preferably 0.01 to 200 mass %, and most preferably 0.1 to 30 mass%, based on the mass of the optically active polymer compound supportedon a carrier.

The carrier used in the present invention includes organic porouscarriers and inorganic porous carriers. The inorganic porous carriersare preferable. Suitable examples of the organic porous carrier includepolymer substances including polystyrenes, polyacrylamides,polyacrylates, or the like. Suitable examples of the inorganic porouscarrier include silica, alumina, magnesia, glass, kaolin, titaniumoxide, silicates and hydroxyapatites.

Silica gel is a particularly preferable carrier. The silica gel has aparticle diameter of 0.1 μm to 10 mm, preferably 1 μm to 300 μm, andmost preferably 1 to 100 μm, and an average pore diameter of 10 Å to 100μm, and preferably 50 to 50,000 Å. Surface treatment may be applied tothe surface of silica gel in order to eliminate effects of residualsilanol, but there is no problem even though the surface treatment isnot applied to the surface.

The solvent used in the present invention may be any generally-usedorganic solvent so long as it can dissolve the optically active polymercompound and the compound having the asymmetric structure of themolecular weight of 1,000 or less.

Examples of the solvent include ketone solvents such as acetone, ethylmethyl ketone or acetophenone; ester solvents such as ethyl acetate,methyl acetate, propyl acetate, methyl propionate or phenyl acetate;ether solvents such as tetrahydrofuran, 1,4-dioxane, diethyl ether,tert-butyl methyl ether or anisole; amide solvents such asN,N-dimethylformamide; imide solvents such asN,N-dimethylimidazolidinone; halogen solvents such as chloroform,methylene chloride, carbon tetrachloride, 1,2-dichloroethane orpentafluoroethanol; hydrocarbon solvents such as pentane, petroleumether, hexane, heptane, octane, benzene, toluene, xylene or mesitylene;alcohol solvents such as methanol, ethanol, propanol or butanol; acidsolvents such as acetic acid, trifluoroacetic acid or formic acid;phenol solvent such as phenol or catechol; and amine solvents such asdiethylamine, triethylamine, pyridine or aniline.

In preparing the optically active polymer compound and the compoundhaving the asymmetric structure of the molecular weight of 1,000 or lessusing such a solvent, a solution concentration is not particularlylimited, and is determined considering ease of contact-supportingtreatment with the carrier, and removal treatment of a solvent in apost-step.

A solvent used to support the optically active polymer compound and thecompound having the asymmetric structure of the molecular weight of1,000 or less on the carrier is removed in the next step.

In the solvent removal treatment in this step, the optically activepolymer compound is still directly supported on the carrier, and thecompound having the asymmetric structure of the molecular weight of1,000 or less maintains the state of being indirectly supported on thecarrier.

In the next step, the optically active polymer compound and the compoundhaving the asymmetric structure of the molecular weight of 1,000 or lessare washed in the state where they are supported on the carrier, therebyremoving the compound having the asymmetric structure of the molecularweight of 1,000 or less.

The washing step can adopt, for example, a method of refluxing under anytemperature of 0° C. to a reflux temperature using acetonitrile,alcohol, hexane, a mixed solvent of hexane and alcohol, or the like.

The amount of solvent used in the case of adopting the reflux method isabout 3 to 50 times the mass of the carrier having supported thereon theoptically active polymer compound and the compound having the asymmetricstructure of the molecular weight of 1,000 or less.

A residual amount of the compound having the asymmetric structure of themolecular weight of 1,000 or less after the washing treatment of thisstep is, when finally formed into a separating agent for separatingenantiomeric isomers, preferably 10 mass % or less, more preferably 2mass % or less, and most preferably 0.5 mass % or less, in theseparating agent. The compound having the asymmetric structure of themolecular weight of 1,000 or less may be contained as an impurity if thecontent is less than the above value.

Further, the compound having the asymmetric structure of the molecularweight of 1,000 or less after the washing treatment of this step can besubstantially removed as well.

A separating agent for separating enantiomeric isomers obtainedaccording to the producing method of the present invention includes anoptically active polymer compound supported on a carrier, further, fromwhich a compound having an asymmetric structure of a molecular weight of1,000 or less added as a production raw material and supported isremoved.

In the separating agent for separating enantiomeric isomers obtainedaccording to the producing method of the present invention, the compoundhaving the asymmetric structure of the molecular weight of 1,000 or lessis the compound of the item (I) or (II), and when the enantiomericisomers are separated using the separating agent for separating theenantiomeric isomers, a separation performance based on a separationcoefficient (α) obtained by the following equation:separation coefficient (α)=(holding coefficient of enantiomer heldrelatively strongly)/(holding coefficient of enantiomer held relativelyweakly)holding coefficient (k′)=[(holding time of the enantiomer)−(deadtime)]/(dead time), where, the dead time is set as elution time of tri-tert-butylbenzene,is desirably shown by the following equation (A) or equation (B):α₁/α₂≧1.05 (provided α₂=1.00)  (A)α₁/α₂≧1.05 (provided α₂>1.00)  (B)where α₁: a separation coefficient of the separating agent forseparating the enantiomeric isomers obtained by adding the compoundhaving the asymmetric structure of the molecular weight of 1,000 or lessin a production step, and

α₂: a separation coefficient of the separating agent for separating theenantiomeric isomers obtained without adding the compound having theasymmetric structure of the molecular weight of 1,000 or less in theproduction step, in which α₂=1.00 means that the enantiomeric isomersare not separated al all, and α₂>1.00 means that the enantiomericisomers are separated.

The equation (A) shows α₂=1.00, that is, the fact that separationperformance of a compound that involves absolutely no separation in thecase of using a separating agent for separating enantiomeric isomersobtained without adding a compound having an asymmetric structure of amolecular weight of 1,000 or less in the production step is improved by5% or more in the case of using the separating agent for separatingenantiomeric isomers of the present invention.

The equation (B) shows α₂>1.00, that is, the fact that separationperformance in the case of using a separating agent for separatingenantiomeric isomers obtained without adding a compound having anasymmetric structure of a molecular weight of 1,000 or less in theproduction step is improved by 5% or more in the case of using theseparating agent for separating enantiomeric isomers of the presentinvention. In the present invention, the separation performance definedby the equation (B) is improved by preferably 10% or more, morepreferably 15% or more, and most preferably 20% or more.

The separating agent for separating enantiomeric isomers of the presentinvention can be used as an immobilizing phase for chromatography suchas gas chromatography, liquid chromatography, thin layer chromatography,supercritical chromatography or capillary electrophoresis. Inparticular, it is preferably used as the chiral immobilizing phase forliquid chromatography. It can also be suitably used as an immobilizingphase for continuous liquid preparative chromatography represented by asimulated moving bed chromatography. The enantiomeric isomers can beseparated with good efficiency using such a separating agent of thepresent invention.

INDUSTRIAL APPLICABILITY

The present invention can greatly improve the separation performance ofa separating agent for separating enantiomeric isomers, andparticularly, such a separating agent is suitable as an immobilizingphase for chromatography and an immobilizing phase for continuous liquidpreparative chromatography.

EXAMPLES

The present invention is described in detail based on examples, but thepresent invention is not limited to those examples.

A representation of the compounds described hereinafter is based on anycompound selected from the following compounds 1 to 16. With respect toa compound having an asymmetric structure of a molecular weight of 1,000or less and a compound to be separated, representation of stericconfiguration (S,R), optical activity (D,L) and racemi form is combinedwith each compound number. For example, when the compound 1 has an Sconfiguration, it is expressed as S-1, and when the compound 1 is aracemi form, it is expressed as racemi form-1.

Example 1

(1) Surface Treatment of Carrier (Silica Gel)

Porous silica gel (particle diameter: 7 μm, micropore: 1,000 Å) wasreacted with 3-aminopropyltriethoxysilane using any conventional methodto perform aminopropylsilane treatment.

(2) Synthesis of Optically Active Polymer Compound

15.0 g of lithium chloride in an absolute dry state was dissolved in 150ml of N,N-dimethylacetamide (DMAc) to prepare a DMAc/LiCl solution.

In a nitrogen atmosphere, 150 ml of the above DMAc/LiCl solution and 150ml of pyridine were added to 10.0 g of cellulose, and the resultingmixture was immersed in an oil bath at 100° C. and stirred for 24 hours.Thereafter, 50 g of 4-methylbenzoyl chloride was added to the mixture toconduct a reaction at 100° C. for 16 hours.

The reaction liquid was added dropwise to 2 L of methanol, followed byreprecipitation and centrifugal separation, thereby obtaining theobjective cellulose tris(4-methylbenzoate) represented by the followingformula.

(3) Preparation of Separating Agent for Separating Enantiomeric Isomers

0.8 g of cellulose tris(4-methylbenzoate) obtained in (2) above and506.0 mg of a compound (S-1) (2-fold molar equivalent to glucose unit ofcellulose tris(4-methylbenzoate)) were dissolved in methylene chlorideto prepare a dope. This dope was applied to 3.2 g of silica gel obtainedin (1) above. After the application, methylene chloride was distilledoff to obtain the objective separating agent for separating enantiomericisomers. This separating agent was added to a mixed solvent ofn-hexane/2-propanol, and the resulting mixture was well stirred, andfiltered. The filtrate was condensed to recover 495.6 mg of (S-1).

(4) Preparation of Packed Column for HPLC

A stainless steel-made column having a length of 25 cm and an innerdiameter of 0.46 cm was packed with the separating agent for separatingenantiomeric isomers obtained in (3) above by a slurry packing methodusing a mixed solvent of n-hexane/2-propanol to obtain a separationcolumn for enantiomeric isomers.

S-1 residual amount in the separating agent for separating enantiomericisomers: 506.0-495.6=10.4 mg

Elution rate of S-1: 495.6/506.0×100=97.9%

S-1 residual amount in the separating agent of the separation column forenantiomeric isomers: (506.0-495.6)/4000×100=0.26%

Example 2

(1) Surface Treatment of Carrier (Silica Gel)

The silica gel treated with aminopropylsilane was obtained in the samemanner as in Example 1.

(2) Synthesis of Optically Active Polymer Compound

In a nitrogen atmosphere, 10.0 g of amylose was added to 300 ml ofpyridine. The resulting mixture was immersed in an oil bath at 100° C.,and 50 g of (S)-phenylethylisocyanate was added thereto to conduct areaction at 100° C. for 48 hours. The reaction liquid was added dropwiseto 2 L of methanol, followed by reprecipitation and centrifugalseparation, thereby obtaining the objective amylosetris[(S)-phenylethylcarbamate] represented by the following equation.

(3) Preparation of Separating Agent for Separating Enantiomeric Isomers

0.8 g of amylose tris[(S)-phenylethylcarbamate] obtained in (2) aboveand 435.65 mg of (S-1) (2-fold molar equivalent to a glucose unit ofamylose tris[(S)-phenylethylcarbamate]) were dissolved in THF to preparea dope. This dope was applied to 3.2 g of silica gel obtained in (1)above. After the application, THF was distilled off to obtain theobjective separating agent for separating enantiomeric isomers. Thisseparating agent was added to a mixed solvent of n-hexane/2-propanol,and the resulting mixture was well stirred, and filtered. The filtratewas condensed to recover 416.0 mg of (S-1).

(4) Preparation of Packed Column for HPLC

A separation column for enantiomeric isomers was obtained in the samemanner as in Example 1.

S-1 residual amount in separating agent for separating enantiomericisomers: 19.6 mg

Elution rate of S-1: 95.5%

S-1 residual amount in separating agent of separation column forenantiomeric isomers: 0.49%

Example 3

(1) Surface Treatment of Carrier (Silica Gel)

The silica gel treated with aminopropylsilane was obtained in the samemanner as in Example 1.

(2) Synthesis of Optically Active Polymer Compound

The objective cellulose tris(3,5-dimethylphenylcarbamate) represented bythe following formula was obtained in the same manner as in Example 2.

(3) Preparation of Separating Agent for Separating Enantiomeric Isomers

0.8 g of cellulose tris(3,5-dimethylphenylcarbamate) obtained in (2)above and 435.9 mg of (S-1) (2-fold molar equivalent for a glucose unitof cellulose tris(3,5-dimethylphenylcarbamate) were dissolved in acetoneto prepare a dope. This dope was applied to 3.2 g of silica gel obtainedin (1) above. After the application, acetone was distilled off to obtainthe objective separating agent for separating enantiomeric isomers. Thisseparating agent was added to a mixed solvent of n-hexane/2-propanol,and the resulting mixture was well stirred, and filtered. The filtratewas condensed to recover 420.6 mg of (S-1).

(4) Preparation of Packed Column for HPLC

A separation column for enantiomeric isomers was obtained in the samemanner as in Example 1.

S-1 residual amount in separating agent for separating enantiomericisomers: 15.3 mg

Elution rate of S-1: 96.5%

S-1 residual amount in separating agent of separation column forenantiomeric isomers: 0.38%

Example 4

(1) Surface Treatment of Carrier (Silica Gel)

The silica gel treated with aminopropylsilane was obtained in the samemanner as in Example 1.

(2) Synthesis of Optically Active Polymer Compound

The objective amylose tris(3,5-dimethylphenylcarbamate) represented bythe following formula was obtained in the same manner as in Example 2.

(3) Preparation of Separating Agent for Separating Enantiomeric Isomers

0.8 g of amylose tris(3,5-dimethylphenylcarbamate) obtained in (2) aboveand 436.3 mg of (S-1) were dissolved in ethyl acetate to prepare a dope.This dope was applied to 3.2 g of silica gel obtained in (1) above.After the application, ethyl acetate was distilled off to obtain theobjective separating agent for separating enantiomeric isomers. Thisseparating agent was added to a mixed solvent of n-hexane/2-propanol,and the resulting mixture was well stirred, and filtered. The filtratewas condensed to recover 427.6 mg of (S-1).

(4) Preparation of Packed Column for HPLC

A separation column for enantiomeric isomers was obtained in the samemanner as in Example 1.

S-1 residual amount in separating agent for separating enantiomericisomers: 8.7 mg

Elution rate of S-1: 98%

S-1 residual amount in separating agent of separation column forenantiomeric isomers: 0.22%

Examples 5 to 24

Using a compound having an asymmetric structure of a molecular weight of1,000 or less shown in Table 1, the objective separating agent forseparating enantiomeric isomers was obtained by the same productionmethod of each of Examples 1 to 3, and thereafter a separation columnfor enantiomeric isomers was obtained.

TABLE 1 Kind of compound having Production method asymmetric structureof (selected from molecular weight of Example Examples 1 to 3) 1,000 orless  5 2 S-3  6 2 D-6  7 2 L-6  8 2 S-7  9 2 R-8 10 2 S-8 11 2 S-9 12 3L-6 13 2  S-11 14 2 R-9 15 1 R-3 16 1 S-3 17 1 D-6 18 1 R-8 19 1 Racemiform-13 20 1 Racemi form-5  21 1 Racemi form-2  22 1 Racemi form-14 23 1Racemi form-15 24 1 Racemi form-16

Comparative Example 1

The objective separating agent for separating enantiomeric isomers wasobtained in the same manner as in Example 1, and thereafter a separationcolumn for enantiomeric isomers was obtained. However, (S-1) was notadded.

Comparative Example 2

The objective separating agent for separating enantiomeric isomers wasobtained in the same manner as in Example 2, and thereafter a separationcolumn for enantiomeric isomers was obtained. However, (S-1) was notadded.

Comparative Example 3

The objective separating agent for separating enantiomeric isomers wasobtained in the same manner as in Example 3, and thereafter a separationcolumn for enantiomeric isomers was obtained. However, (S-1) was notadded.

Comparative Example 4

The objective separating agent for separating enantiomeric isomers wasobtained in the same manner as in Example 4, and thereafter a separationcolumn for enantiomeric isomers was obtained. However, (S-1) was notadded.

Application Examples 1 to 45

Using the separation columns for enantiomeric isomers obtained inExamples 1 to 24 and Comparative Examples 1 to 4, a values were measuredby liquid chromatography (liquid chromatograph, manufactured by JASCOCo.). The measurement conditions include moving phase:n-hexane/2-propanol=90/10, flow rate: 1.0 ml/min, temperature: 25° C.,and detection wavelength: 254 nm. The results are shown in Tables 2 to4.

The judgement in the tables is judgement of identity, similarity ornon-similarity, and the details thereof are described after the tables.An increase rate (%) of a value in the tables was obtained by thefollowing equation: α₁−α₂/α₁×100. Here, expressed by α=1.00 is the statewhere asymmetry identification was not conducted at all and only onepeak was observed.

TABLE 2 Kind of Kind of Kind of compound having separation separationcolumn Increase asymmetric structure of Compound column and α value rateof Application molecular weight of to be separated and α value Com. αvalue example 1,000 or less Kind Judgment Ex. α₁ Ex. α₂ (%) 1 S-1 Racemiform-1 Identical 1 1.11 1 1.00 11 2 S-1 Racemi form-1 Identical 2 4.24 23.73 14 3 S-1 Racemi form-1 Identical 3 1.07 3 1.00 7 4 S-1 Racemiform-2 Similar 4 1.05 4 1.00 5 5 S-3 Racemi form-4 Similar 5 2.76 2 2.3219 6 S-3 Racemi form-5 Similar 5 3.51 2 2.45 43 7 D-6 Racemi form-5Similar 6 3.13 2 2.45 28 8 L-6 Racemi form-4 Similar 7 2.78 2 2.32 20 9L-6 Racemi form-5 Similar 7 3.78 2 2.45 54 10 S-7 Racemi form-2 Similar8 1.17 2 1.00 17 11 S-7 Racemi form-5 Similar 8 3.06 2 2.45 25 12 R-8Racemi form-2 Similar 9 1.29 2 1.00 29 13 R-8 Racemi form-4 Similar 92.68 2 2.32 16 14 S-8 Racemi form-2 Similar 10 1.22 2 1.00 22 15 S-8Racemi form-5 Similar 10 3.11 2 2.45 27 16 S-9 Racemi form-4 Similar 112.79 2 2.32 20 17 S-9 Racemi form-5 Similar 11 2.73 2 2.45 11 18 S-1Racemi form-2 Similar 2 1.12 2 1.00 12 19 S-1 Racemi form-5 Similar 22.90 2 2.45 18

Application Examples 4 and 18: The compound having the asymmetricstructure and the compound to be separated each have an asymmetriccarbon atom at the root of a phenyl group. Further, adjacent carbonatoms (β-position) of the asymmetric carbon atoms each have a carbonylgroup, and the environment around the asymmetric carbon atoms issimilar.

Application Examples 5, 8, 16 and 19: The compound having the asymmetricstructure and the compound to be separated each have an asymmetriccarbon atom at the root of a hydroxyl group. Further, the environmentaround the asymmetric carbon atom is similar.

Application Examples 6, 7 and 9: The compound having the asymmetricstructure and the compound to be separated each have an asymmetriccarbon atom at the root of a hydroxyl group. Further, adjacent carbonatoms (α-position) of the asymmetric carbon atoms are each carbonylgroups, and thus, the environment around the asymmetric carbon atoms issimilar.

Application Examples 10, 12 and 14: The compound having the asymmetricstructure and the compound to be separated each have an asymmetriccarbon atom adjacent to an oxygen atom α-position). Further, adjacentcarbon atoms (β-position) of the asymmetric carbon atoms each have acarbonyl group, and the environment around the asymmetric carbon atomsis similar.

Application Example 11: The compound having the asymmetric structure andthe compound to be separated each have an asymmetric carbon atomadjacent to a carbon atom and have a carbonyl group in a molecule. Thus,the environment around the asymmetric carbon atoms is similar.

Application Example 13: The compound having the asymmetric structure andthe compound to be separated each have an asymmetric carbon atom at theroot of a trihalogenomethyl substituent. Further, the environment aroundthe asymmetric carbon atom is similar.

Application Examples 15 and 17: The compound having he asymmetricstructure and the compound to be separated each have an asymmetriccarbon atom adjacent to an oxygen atom (α-position) and have a carbonylgroup in the vicinity of the asymmetric carbon atoms, and theenvironment around the asymmetric carbon atoms is similar.

TABLE 3 Kind of compound having Kind of Kind of Increase asymmetricstructure of separation column separation column rate of Applicationmolecular weight of Compound to be separated and α value and α value αvalue example 1,000 or less Kind Judgment Ex. α₁ Com. Ex. α₂ (%) 20 L-6Racemi form-10 Non-similar 12 2.68 3 2.43 10 21 S-11 Racemi form-5Non-similar 13 2.92 2 2.45 19 22 S-3 Racemi form-10 Non-similar 5 1.73 21.38 25 23 S-3 Racemi form-2 Non-similar 5 1.26 2 1.00 26 24 S-3 Racemiform-12 Non-similar 5 1.98 2 1.71 16 25 D-6 Racemi form-10 Non-similar 61.54 2 1.38 12 26 D-6 Racemi form-2 Non-similar 6 1.21 2 1.00 21 27 L-6Racemi form-10 Non-similar 7 1.71 2 1.38 24 28 L-6 Racemi form-2Non-similar 7 1.31 2 1.00 31 29 L-6 Racemi form-12 Non-similar 7 1.98 21.71 16 30 R-8 Racemi form-5 Non-similar 9 3.69 2 2.45 51 31 R-8 Racemiform-12 Non-similar 9 2.01 2 1.71 18 32 S-8 Racemi form-5 Non-similar 103.11 2 2.45 27 33 R-9 Racemi form-2 Non-similar 14 1.06 2 1.00 6 34 S-9Racemi form-2 Non-similar 11 1.08 2 1.00 8 35 R-3 Racemi form-12Non-similar 15 1.49 1 1.22 22 36 S-3 Racemi form-12 Non-similar 16 1.531 1.22 25 37 D-6 Racemi form-12 Non-similar 17 1.48 1 1.22 21 38 R-8Racemi form-4 Non-similar 18 1.50 1 1.35 11

TABLE 4 Kind of Kind of compound having Kind of separation columnIncrease asymmetric structure of separation column and α value rate ofApplication molecular weight of Compound to be separated and α valueCom. α value example 1,000 or less Kind Judgment Ex. α₁ Ex. α₂ (%) 39Racemi form-13 Racemi form-13 Identical 19 1.82 1 1.67 9 40 Racemiform-14 Racemi form-14 Similar 19 1.61 1 1.35 19 41 Racemi form-5 Racemiform-12 Non-similar 20 1.41 1 1.22 16 42 Racemi form-2 Racemi form-12Non-similar 21 1.52 1 1.22 25 43 Racemi form-14 Racemi form-12Non-similar 22 1.48 1 1.22 21 44 Racemi form-15 Racemi form-12Non-similar 23 1.46 1 1.22 20 45 Racemi form-16 Racemi form-12Non-similar 23 1.43 1 1.22 17

Application Example 40: The compound having the asymmetric structure andthe compound to be separated each have an asymmetric carbon atom at theroot of a hydroxyl group, and the structure of the molecule as a wholeincludes a condensed ring similar to a naphthyl group and an anthranylgroup. Therefore, the environment around the asymmetric carbon atom issimilar.

1. A separating agent for separating enantiomeric isomers, comprising anoptically active polymer compound supported on a carrier, formed by aprocess, comprising a step of supporting an optionally active polymercompound and a compound having asymmetric structure of a molecularweight of 1,000 or less on a carrier using a solvent added as aproduction raw material; a step of removing the solvent; and a step ofremoving the compound having the asymmetric structure of the molecularweight of 1,000 or less by washing to form the separating agent havingenhanced asymmetric identification ability.
 2. The separating agent forseparating enantiomeric isomers as claimed in claim 1, wherein aresidual amount of the compound having the asymmetric structure of themolecular weight of 1,000 or less is 10 mass % or less in the separatingagent for separating the enantiomeric isomers.
 3. The novel separatingagent for separating enantiomeric isomers as claimed in claim 1, whereinthe compound having the asymmetric structure of the molecular weight of1,000 or less is substantially removed.
 4. The novel separating agentfor separating enantiomeric isomers as claimed in claim 1, whereinsupporting the optically active polymer compound on the carrier isconducted by physical adsorption.
 5. The novel separating agent forseparating enantiomeric isomers as claimed in claim 1, whereinsupporting the optically active polymer compound on the carrier isconducted by chemical bonding.
 6. The novel separating agent forseparating enantiomeric isomers as claimed in claim 5, wherein thecarrier and the optically active polymer compound are chemically bondedbefore or after the removal of the compound having the asymmetricstructure of the molecular weight of 1,000 or less.
 7. The novelseparating agent for separating enantiomeric isomers as claimed in claim5, wherein the carrier and the optically active polymer compound arechemically bonded by a reaction with a crosslinking agent, a reactionwith a radical generator or irradiation with light before or after theremoval of the compound having the asymmetric structure of the molecularweight of 1,000 or less.
 8. The separating agent for separatingenantiomeric isomers as claimed in claim 1, wherein the optically activepolymer compound has an asymmetry identification ability to a compoundto be separated when used as the separating agent for separating theenantiomeric isomers.
 9. The separating agent for separatingenantiomeric isomers as claimed in claim 1, wherein the optically activepolymer compound is a polysaccharide derivative.
 10. The separatingagent for separating enantiomeric isomers as claimed in claim 1, whereinthe optically active polymer compound is a polysaccharide esterderivative or carbamate derivative.
 11. The separating agent forseparating enantiomeric isomers as claimed in claim 1, wherein theoptically active polymer compound is a cellulose derivative or anamylose derivative.
 12. The separating agent for separating enantiomericisomers as claimed in claim 1, wherein the optically active polymercompound is a polymer or a copolymer of acrylamides, methacrylamides,acrylates or methacrylates having an optically active substituent on aside chain.
 13. The separating agent for separating enantiomeric isomersas claimed in claim 1, wherein an amount of the optically active polymercompound supported on the carrier is 1 to 100 mass % to a mass of thecarrier.
 14. The separating agent for separating enantiomeric isomers asclaimed in claim 1, wherein the compound having the asymmetric structureof the molecular weight of 1,000 or less is a compound to be separatedor a similarly structured compound thereof when used as the separatingagent for separating the enantiomeric isomers.
 15. The separating agentfor separating enantiomeric isomers as claimed in claim 1, wherein thecompound having the asymmetric structure of the molecular weight of1,000 or less is not the compound to be separated or a similarlystructured compound thereof when used as the separating agent forseparating the enantiomeric isomers.
 16. The novel separating agent forseparating enantiomeric isomers as claimed in claim 15, wherein thecompound having the asymmetric structure of the molecular weight of1,000 or less is not the compound to be separated or a similarlystructured compound thereof when used as the separating agent forseparating the enantiomeric isomers, and is a compound having a cyclicstructure of a molecular weight of 40 to
 1000. 17. The novel separatingagent for separating enantiomeric isomers as claimed in claim 1, whereinthe compound having the asymmetric structure of the molecular weight of1,000 or less is a racemic form and/or an optically active substance.18. The separating agent for separating enantiomeric isomers as claimedin claim 1, wherein the amount of the compound having the asymmetricstructure of the molecular weight of 1,000 or less supported on thecarrier is 0.01 to 1,000 mass % to a mass of the optically activepolymer compound.
 19. The novel separating agent for separatingenantiomeric isomers as claimed in claim 1, wherein the compound havingthe asymmetric structure of the molecular weight of 1,000 or less is acompound to be separated or a similarly structured compound thereof whenused as a separating agent for separating enantiomeric isomers, and whenthe enantiomeric isomers are separated using the separating agent forseparating the enantiomeric isomers, a separation performance based on aseparation coefficient (α) obtained by the following equation:separation coefficient (α)=(holding coefficient of enantiomer heldrelatively strongly)/(holding coefficient of the enantiomer heldrelatively weakly)holding coefficient (k′)=[(holding time of the enantiomer)−(deadtime)]/(dead time) is represented by the following equation (A) orequation (B):α₁/α₂≧1.05 (provided α₂=1.00)  (A)α₁/α₂≧1.05 (provided α₂>1.00)  (B) where α₁: a separation coefficient ofthe separating agent for separating the enantiomeric isomers obtained byadding the compound having the asymmetric structure of the molecularweight of 1,000 or less in a production step, and α₂: a separationcoefficient of the separating agent for separating the enantiomericisomers obtained without adding the compound having the asymmetricstructure of the molecular weight of 1,000 or less in the productionstep, in which α₂=1.00 means that the enantiomeric isomers are notseparated al all, and α₂>1.00 means that the enantiomeric isomers areseparated.
 20. The novel separating agent for separating enantiomericisomers as claimed in claim 1, wherein the compound having theasymmetric structure of the molecular weight of 1,000 or less is not thecompound to be separated or the similarly structured compound thereofwhen used as the separating agent for separating the enantiomericisomers, and when the enantiomeric isomers are separated using theseparating agent for separating the enantiomeric isomers, a separationperformance based on a separation coefficient (α) obtained by thefollowing equation:separation coefficient (α)=(holding coefficient of enantiomer heldrelatively strongly)/(holding coefficient of the enantiomer heldrelatively weakly)holding coefficient (k′)=[(holding time of the enantiomer)−(deadtime)]/(dead time) is represented by the following equation (A) orequation (B):α₁/α₂≧1.05 (provided α₂=1.00)  (A)α₁/α₂≧1.05 (provided α₂>1.00)  (B) where α₁: a separation coefficient ofthe separating agent for separating the enantiomeric isomers obtained byadding the compound having the asymmetric structure of the molecularweight of 1,000 or less in a production step, and α₂: a separationcoefficient of the separating agent for separating the enantiomericisomers obtained without adding the compound having the asymmetricstructure of the molecular weight of 1,000 or less in the productionstep, in which α₂=1.00 means that the enantiomeric isomers are notseparated al all, and α₂>1.00 means that the enantiomeric isomers areseparated.
 21. The separating agent for separating enantiomeric isomersas claimed in claim 20, wherein the compound having the asymmetricstructure of the molecular weight of 1,000 or less is not a compound tobe separated or a similarly structured compound thereof when used as theseparating agent for separating the enantiomeric isomers, and is acompound having a cyclic structure of a molecular weight of 40 to 1000.22. An immobilizing phase for a chromatography using the separatingagent for separating enantiomeric isomers as claimed in claim
 1. 23. Animmobilizing phase for a continuous liquid preparative chromatographyusing the separating agent for separating enantiomeric isomers asclaimed in claim
 1. 24. A separation method for enantiomeric isomersusing the separating agent for separating the enantiomeric isomers asclaimed in claim 1.